We study the molecular mechanisms involved in assembly and function of translation initiation complexes involved in protein synthesis, using yeast as a model system to exploit its powerful combination of genetics and biochemistry for dissecting complex cellular processes in vivo. The translation initiation pathway produces an 80S ribosome bound to mRNA with methionyl initiator tRNA (tRNAi) base-paired to the AUG start codon. The tRNAi is recruited to the 40S subunit in a ternary complex (TC) with GTP-bound eIF2 to produce the 43S preinitiation complex (PIC) in a reaction stimulated by eIFs 1, 1A, 3 and 5. The 43S PIC attaches to the 5' end of mRNA, facilitated by cap-binding complex eIF4F (comprised of eIF4E, eIF4G, and RNA helicase eIF4A) and PABP bound to the poly(A) tail, and scans the 5 untranslated region (UTR) for the AUG start codon. Scanning is promoted by eIFs 1 and 1A, which induce an open conformation of the 40S and TC binding in a conformation suitable for scanning successive triplets entering the ribosomal P site (P-out), and by eIF4F and other RNA helicases, such as Ded1, that remove secondary structure in the 5' UTR. AUG recognition leads to tighter binding of TC in the P-in state and evokes irreversible hydrolysis of the GTP bound to eIF2, dependent on the GTPase activating protein (GAP) eIF5, releasing eIF2-GDP from the PIC leaving tRNAi in the P site. After joining of the 60S subunit, producing the 80S initiation complex, the eIF2-GDP is recycled to eIF2-GTP by guanine nucleotide exchange factor (GEF) eIF2B for the next round of translation initiation. Interface between 40S exit channel protein uS7/Rps5 and eIF2 modulates start codon recognition in vivo Our recent cryo-EM analysis of partial yeast PICs revealed distinct conformations relevant to different stages of initiation. A py48S-open complex exhibits upward movement of the 40S head from the body that widens both the mRNA binding cleft and the P site and eliminates interactions of the 40S subunit with Met-tRNAi and mRNA that are evident in the py48S-closed complex. The py48S-open conformation seems well-suited for scanning of successive triplets for complementarity to Met-tRNAi, with TC anchored in the unstable P-out conformation; whereas py48S-closed exhibits the more stable P-in conformation required for start codon selection. During the transition from py48S-open to py48S-closed, domain 1 (D1) of the alpha subunit of eIF2 rotates slightly to avoid a clash with the 40S body, which alters the interface between eIF2a-D1 and the C-terminal helix of the 40S protein uS7/Rps5. Certain eIF2/uS7 contacts appear to be enhanced in the open conformation (D77-R219 & D84-S223) and thus might be expected to promote scanning through UUG or poor-context AUG codons. A third contact (Y82-D215) is favored in the closed conformation and might have the opposite effect of enabling recognition of suboptimal initiation sites by favoring the more stable P-in conformation. We made amino acid substitutions that perturb these contacts and determined their effects on initiation in vivo and stability of TC binding to reconstituted PICs in vitro. uS7 substitutions disrupting eIF2 contacts favored in the open complex were found to increase initiation at near-cognate UUG codons and AUGs in poor Kozak context; and among these, uS7-S223D was shown to stabilize TC binding to PICs reconstituted with a UUG-containing mRNA. Conversely, uS7-D215 substitutions, perturbing uS7-eIF2 interaction only in the closed state, conferred the opposite phenotypes of diminished initiation at UUG and poor AUG codons and (for D215L) accelerated TC dissociation from reconstituted PICs. Our results support the physiological importance of the differential contacts between uS7 and eIF2-D1 in py48S-open versus py48S-closed structures and demonstrate that different conformations of the uS7/eIF2 interface stabilize first the open, and then the closed state of the PIC to promote accurate AUG selection in vivo. Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons eIF3 is a multisubunit complex that promotes binding of TC and mRNA to the PIC, and enhances scanning and accurate start codon recognition. Our recent structural analysis revealed that yeast eIF3 encircles the PIC and interacts with the mRNA-entry and -exit channel pores. We recently presented evidence that the eIF3a subunit stabilizes interaction between the PIC and mRNA at the exit channel in a manner that is functionally redundant with mRNA:PIC interactions at the entry channel. The cryo-EM structure of py48S-closed revealed contacts between mRNA in the entry channel and amino acids of 40S protein uS3/Rps3. We have now shown that substitutions of these uS3 residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons; and that two such substitutions, R116D and R117D, also increase discrimination against an AUG codon in poor context. Consistently, R116D and R117D destabilized TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, indicating destabilization of the P-in state with a UUG:anticodon mismatch. Using model mRNAs designed to lack contacts with either the mRNA entry or exit channels of the 40S subunit, we further demonstrated that Arg116/Arg117 are crucial for stabilizing PIC:mRNA contacts specifically at the entry channel, thus complementing the function of eIF3 at the exit channel. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life. eIF4B and DEAD-box RNA helicases Ded1 and eIF4A preferentially stimulate translation of long mRNAs with structured 5UTRs and low closed-loop potential. RNA helicases eIF4A and Ded1 are believed to resolve mRNA secondary structures that impede ribosome attachment to the mRNA and scanning to the start codon, but whether they perform distinct functions in vivo was poorly understood. We recently compared the effects of mutations in Ded1 or eIF4A on translational efficiencies (TEs) by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of Ded1 substantially reduced the relative TEs of >600 mRNAs, whereas inactivation of eIF4A similarly affected far fewer (<40) mRNAs. Ded1-dependent mRNAs show greater than average 5' UTR length and secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Whereas Ded1 is required for scanning through secondary structures, it appears that eIF4A promotes a distinct step of initiation equally important for all mRNAs, such as ribosome attachment. eIF4B is regarded as a cofactor for eIF4A; however, ribosome profiling of a mutant lacking eIF4B indicated a reduction in relative TE for many more mRNAs than were affected on inactivation of eIF4A, supporting an eIF4A-independent role for eIF4B. Interestingly, eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a manner mitigated by their ability to form closed-loop' circular mRNPs via eIF4F-Pab1 association, thus suggesting cooperation between closed-loop assembly and eIF4B and helicase functions in stimulating translation initiation in vivo.